Nucleic acid construct for expressing active substances which can be activated by proteases, and preparation and use

ABSTRACT

The invention relates to a nucleic acid construct for expressing an active substance which is activated by an enzyme which is released from mammalian cells, which construct comprises the following components: a) at least one promoter element, b) at least one DNA sequence which encodes an active compound (protein B), c) a least one DNA sequence which encodes an amino acid sequence (part structure C) which can be cleaved specifically by an enzyme which is released from a mammalian cell, and d) at least one DNA sequence which encodes a peptide or protein (part structure D) which is bound to the active compound (protein B) by way of the cleavable amino acid sequence (part structure C) and inhibits the activity of the active compound (protein B), and also to the use of the nucleic acid construct for preparing a drug for treating diseases.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a nucleic acid construct forexpressing active substances which can be activated by proteases and toits preparation and use.

[0002] Like inflamed areas, tumors are distinguished from thesurrounding normal tissue by a substantial increase in the formation andsecretion of proteases [Schmitt et al., Fibrinol. 6, 3 (1992), Cottam etal., Int. J. Oncol. 2, 861 (1993), Tryggvason et al., Breast Cancer Res.And Treatm. 24, 209 (1993), Leto et al., Anticancer Res. 12, 235 (1992),Hart, Fibrinol. 6, 11 (1992), Albini et al., J. Natl. Cancer Inst. 83,735 (1991)]. Examples of these proteases are plasminogen activators,cathepsins and matrix metalloproteinases.

[0003] An essential function of these tumor proteases is to dissolve theextracellular matrix to allow the tumor cells to invade, and grow in aninfiltrative manner in, normal tissue. At the same time, these proteasesprotect the tumor from the defence mechanisms of the body insofar as theactive compounds which are required for defence are cleaved, and therebyinactivated, by the proteases which are formed by the tumor. Thus, forexample, antibodies, cytokines and growth factors, complement factors,coagulation factors and mediators are inactivated by tumor proteases.

[0004] In the past, the aim was, therefore, to inhibit the infiltrativegrowth and metastatic growth of tumors, and inactivation of the defencemechanisms of the body, by inhibiting the tumor cell proteases [Hocman,Int. J. Biochem. 24, 1365 (1992), Troll et al., JNCI 73, 1245 (1984),Ray et al., Eur. Respir. 7, 2062 (1994), Koop et al., Cancer Res. 54,4791 (1994), Chiriri et al., Int. J. Cancer 58, 460 (1994), Denhardt etal., 59, 329 (1993), Melchiori et al., Cancer Res. 52, 2353 (1992)].However, particularly for stoichiometric and pharmacokinetic reasons,little success has previously been achieved in inhibiting tumor cellproteases.

[0005] An attempt was therefore made to use the tumor cell proteases toactivate bacterial toxins such as Staphylococcus aureus α-hemolysin[Panchal et.al., Nature Biotechn. 14, 852 (1996)]. For this, an aminoacid sequence, i.e. XX-Arg-X, was inserted into positions 129 to 132 ofthe α-hemolysin and in this way inactive mutants were produced which areonly cleaved, and thereby activated, by tumor proteases such ascathepsin B.

[0006] Based on these results, proimmunolysins were proposed [Panchal etal., Nature Biotechn. 14, 852 (1996)], which proimmunolysins comprise anantibody which is coupled to a Staphylococcus aureus α-hemolysin whichcan be activated by tumor proteases or to a sea anemone equinatoxin II,with the antibody determining the target cell specificity of thecoupling product.

[0007] However, the proposed concept suffers from the followingdisadvantages in relation to its use in tumor therapy:

[0008] In the first place, the authors chose xenogeneic nonendogenouslysins and/or toxins which are immunogenic for the host organism (forexample, patients) and as a result induce an immune reaction in the hostorganism, which immune reaction neutralizes and inactivates theantibody/toxin conjugate. In the second place, it is known [Sedlacek etal., Antibodies as Carriers of Cytotoxicity, Contrib. to Oncol. 43,Karger Verlag, Munich, 1992] that, due to their molecular size and tothe Theological conditions at the tumor, tumor-specific antibodies andimmunotoxins only accrue in very small quantities (0.01-0.001% of thegiven antibody or immunotoxin/g of tumor) at the tumor and onlypenetrate the tumor to an incomplete extent so that it is either notpossible to destroy all the tumor cells or only possible to destroy asmall portion of the cells of a tumor. Then again, the extent to whichtumor antigens, against which the antibody is directed, are expressedusually differs between the individual tumor cells, and the variable,antigen-negative tumor cells readily evade the attack by the antibodiesor the immunotoxins. In addition to this, antigens which are secreted bythe tumor cells neutralize the antibodies at the periphery of the tumor(Sedlacek et al., Monoclonal Antibodies in Tumor Therapy, Contrib. toOncol., Karger Verlag, 1988).

[0009] Consequently, there is still a great need for a targetcell-specific therapy for tumors and inflammations.

SUMMARY OF THE INVENTION

[0010] An object of the present invention is therefore to provide anactive compound against tumors and inflammations, which active compounddoes not exhibit said disadvantages. The present invention thereforerelates to a novel technique which uses the secretion of enzymes intumors or areas of inflammation to achieve the local release of activecompounds whose inactive precursors are expressed in tumor cells,tumor-associated cells or inflammatory cells.

[0011] One part of the subject-matter of the present invention istherefore a nucleic acid construct for expressing an active substancewhich is activated by an enzyme which is released from mammalian cells,which nucleic acid construct comprises the following components:

[0012] a) at least one promoter element,

[0013] b) at least one DNA sequence which encodes an active compound(protein B),

[0014] c) at least one DNA sequence which encodes an amino acid sequence(part structure C) which can be cleaved specifically by an enzyme whichis released from a mammalian cell, and

[0015] d) at least one DNA sequence which encodes a peptide or protein(part structure D) which is bound to the active compound (protein B) byway of the cleavable amino acid sequence (part structure C) and inhibitsthe activity of the active compound (protein B).

[0016] Further objects of the instant invention are described asfollows:

[0017] 1. A nucleic acid construct for expressing an active substancewhich is activated by an enzyme which is released from mammalian cells,wherein the construct comprises the following nucleic acid sequences inthe following order:

[0018] a) at least one promoter element operably linked to;

[0019] b) at least one nucleic acid sequence which encodes an activecompound, wherein the active compound is endogenous to mammals, operablylinked to;

[0020] c) at least one nucleic acid sequence which encodes an amino acidsequence cleavable specifically by an enzyme which is released from amammalian cell, operably linked to;

[0021] d) at least one DNA sequence which encodes a polypeptide which isbound to the active compound by the cleavable amino acid sequence andinhibits the activity of the active compound, and wherein the nucleicacid component c) does not naturally occur as operably linking thenucleic acid sequence b) to the nucleic acid d).

[0022] 2. A nucleic acid construct as described in 1, wherein the enzymeis a protease.

[0023] 3. A nucleic acid construct as described in 1, wherein the enzymeis a prostate specific antigen, a plasminogen activator, a cathepsin ora matrix metalloproteinase.

[0024] 4. A nucleic acid construct as described in 1, wherein themammalian cells are tumor cells, leukemia cells, endothelial cells,macrophages, lymphocytes, muscle cells, epithelial cells, glia cells,synovial cells or virus-infected cells.

[0025] 5. A nucleic acid construct as described in 1, wherein thenucleic acid construct further comprises a nucleic acid sequenceoperably linked to the construct of 1, wherein the nucleic acid sequenceencodes a ligand which binds the active compound to a target structure.

[0026] 6. A nucleic acid construct as described in 1, wherein thenucleic acid sequences b) and d) of 1 encode parts of a naturalprecursor of a protein active compound, wherein the nucleic acidsequence encoding the cleavage sequence naturally occurring between thenucleic acid sequences b) and d) has been replaced by the nucleic acidsequence c), which does not naturally occur between the nucleic acidsequences b) and d).

[0027] 7. A nucleic acid construct as described in 1, wherein thepolypeptide encoded by the nucleic acid sequence d) is part of a naturalprecursor of a protein active compound.

[0028] 8. A nucleic acid construct as described in 1, wherein theconstruct is operably inserted into a plasmid or a viral vector.

[0029] 9. A nucleic acid construct as described in 1, wherein thenucleic acid sequence a) is a promoter sequence which can be activatednonspecifically, cell-specifically, virus-specifically, metabolically,cell cycle-specifically or by tetracycline.

[0030] 10. A nucleic acid construct as described in 1, wherein thenucleic acid sequence a) comprises at least two identical or twodifferent promoter sequences.

[0031] 11. A nucleic acid construct as described 9, wherein the nucleicacid sequence a) is activated in endothelial cells, in cells adjoiningactivated endothelial cells, in muscle cells, in leukemia cells, intumor cells, in glia cells, in lymphocytes, in macrophages or insynovial cells.

[0032] 12. A nucleic acid construct as described in 1, wherein theactive compound activates or inhibits a biological activation cascade oris an active component of this cascade, or activates or inhibits thecoagulation system, activates fibrinolysis, activates the complementsystem or activates the kinin system, or is an enzyme which converts theinactive precursor of a pharmacological substance into the activesubstance, or which itself is a pharmacologically active substance.

[0033] 13. A nucleic acid construct as described in 12, wherein theactive compound is a coagulation factor which is selected from the groupconsisting of thrombin, factor Va, factor VIIa, factor IXa, factor Xa,TF coagulation-active fragments or factor XIIa; thrombin which ismutated in the region of the Arg-Thr cleavage site (amino acid position327/328); a fibrinolytic protein which is selected from urokinase, tPAor functional hybrids thereof; a complement factor which is selectedfrom CVF, C3b or functional cleavage products thereof; an antithromboticprotein which is selected from protein C, C-1S inhibitor,a1-antitrypsin, hirudin, AT-III, TFPI, PAI-1, PAI-2 or PAI-3; akallikrein; a cytostatic, cytotoxic or inflammation-eliciting protein;an antiangiogenic protein; an immunomodulatory protein; anantiinflammatory protein; a protein which relieves damage to the nervoussystem; a protein which inhibits or neutralizes the neurotoxic effect ofTNFα; an angiogenesis-stimulating protein; a hypotensive protein; anantiviral protein; a cytokine; an interferon; a tumor necrosis factor;oncostatin M or LIF; a cytokine receptor; the moiety of a cytokinereceptor which is external to the cell; a cytokine antagonist; a growthfactor; a growth factor receptor; the moiety of a growth factor receptorwhich is external to the cell; a chemokine; angiostatin; platelet factor4; TIMP-1, TIMP-2 or TIMP-3; a nitroreductase; a β-glucuronidase; acarboxypeptidase; a β-lactamase; a cytosine deaminase; a catalase; aperoxidase; a phosphatase; an oxidase; kallikrein or an endothelial cellnitric oxide synthase.

[0034] 14. A nucleic acid construct as described in 1, which furthercomprises a nucleic acid sequence b′) which encodes a ligand which bindsto a cell membrane receptor, a cell membrane antigen, a cellmembrane-located adhesion molecule, or to the extracellular matrix orcomponent thereof.

[0035] 15. A nucleic acid construct as described in 14, wherein theligand is an antibody or an antibody fragment which binds specificallyto a cell membrane antigen or to an antigen on the extracellular matrix,or is a polypeptide which binds to receptor on the cell membrane whereinthe polypeptide is a growth factor, a cytokine, an interferon, a tumornecrosis factor, a chemokine, a receptor-binding part sequence of theseligands, a peptide hormone, angiotensin, kinin, folic acid, an adhesionmolecule or the part sequence of the adhesion molecule which binds tothe corresponding adhesion molecule or to the extracellular matrix, anextracellular moiety of an Fc receptor, a glycoprotein of a virus, apart sequence of the glycoprotein which binds to these cells, thetransmembrane domain of a receptor or of a viral glycoprotein, or aglycophospholipid anchor.

[0036] 16. A nucleic acid construct as described in 14, wherein theligand binds to activated or proliferating endothelial cells, to tumorcells, to muscle cells, preferably smooth muscle cells, to fibroblasts,to macrophages, to lymphocytes, to liver cells, to kidney cells, tosynovial cells, to inflammatory cells, to virus-infected cells, tobronchial epithelial cells, to glia cells or to leukemia cells.

[0037] 17. A nucleic acid construct as described in 14, wherein theconstruct comprises at least two identical or different nucleic acidsequences b)c)d) or b′)b)c)d), which nucleic acid sequences are linkedto each other by way of an internal ribosomal entry site.

[0038] 18. A process for preparing a nucleic acid construct according to1, which comprises operably linking the nucleic acid sequences of 1.

[0039] 19. A method for the treatment or prophylaxis of tumors,leukemias, allergies, autoimmune diseases, infections, inflammations,transplant rejection reactions, thromboses, blood vessel occlusions,blood coagulation, blood circulation disturbances, injuries to tissues,or damage to the nervous system, comprising administering to a mammal aneffective amount of a polypeptide expressed by the nucleic acidconstruct of 1.

[0040] 20. A method for preparing a recombinantly altered cell,comprising transducing a suitable cell with the nucleic acid constructof 1.

[0041] 21. A method for preparing a polypeptide which is encoded by thenucleic acid construct of 1, comprising transducing a suitable cell withthe construct, expressing the polypeptide in the cell, and isolating theexpressed polypeptide.

[0042] 22. The method of 20, wherein the cell is an endothelial cell, alymphocyte, a macrophage, a glia cell, a fibroblast, a liver cell, akidney cell, a muscle cell, a cell of the bone or cartilage tissue, asynovial cell, a peritoneal cell, a skin cell, an epithelial cell, aleukemia cell or a tumor cell.

[0043] 23. The method of 21, wherein the cell is an endothelial cell, alymphocyte, a macrophage, a glia cell, a fibroblast, a liver cell, akidney cell, a muscle cell, a cell of the bone or cartilage tissue, asynovial cell, a peritoneal cell, a skin cell, an epithelial cell, aleukemia cell or a tumor cell.

[0044] 24. A cell transduced with the nucleic acid construct of 1.

[0045] 25. A protein encoded by the nucleic acid construct of 1.

[0046] The term “endogenous to mammals” as used to describe the activecompound of the instant invention denotes a polypeptide that isnaturally expressed in mammals or a derivative thereof as discussedherein.

[0047] The term “does not naturally occur” as used to describe thelinking nucleic acid component c) denotes that the described componentc) of the instant invention is not found in nature as operably linkingcomponents b) and d).

BRIEF DESCRIPTION OF THE DRAWINGS

[0048]FIG. 1 is a diagrammatic representation of a novel nucleic acidconstruct comprising components a), b), c) and d);

[0049]FIG. 2 is a diagrammatic representation of a novel nucleic acidconstruct which has been enlarged by adding component b′); and

[0050]FIG. 3 is diagrammatic representation of a nucleic acid constructfor PSA-activatable factor X.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] In their simplest form, the individual components can bearranged, for example, as shown in FIG. 1. In this case, expression of aprotein BCD, encoded by components b), c) and d), is induced byactivating the promoter sequence [component a)]. The amino acid sequenceC of the expression product is then cleaved by cellular enzymes, e.g.proteases, as a result of which protein B, which constitutes the activecompound, is released. Within the meaning of the present invention,proteases or enzymes are to be understood as being one or more proteasesor enzymes.

[0052] In another embodiment, said enzyme is a protease, in particular aplasminogen activator, a cathepsin or a matrix metalloproteinase. Saidmammalian cells are preferably tumor cells, leukemia cellsi endothelialcells, macrophages, lymphocytes, muscle cells, epithelial cells, gliacells, synovial cells or virus-infected cells.

[0053] Enzymes are preferably released, in an organism, by tumors andtumor cells and also by cells which are involved in an inflammatoryprocess [Barrett et al., Mammalian Proteases, Academic Press, London1980; Sedlacek and Möröy, Immune Reactions, Springer Verlag, 1995)].

[0054] According to the present invention, component c) is consequentlyselected such that the expressed protein, e.g. BCD, is preferablycleaved, in its part structure C, by proteases which are formed intumors or secreted by tumor cells or inflammatory cells. Examples ofthese proteases are plasminogen activators, such as plasminogenactivator of the urokinase type or tissue plasminogen activator;cathepsins, such as cathepsin B, cathepsin D, cathepsin L, cathepsin Eor cathepsin H, or their precursors (procathepsins); matrixmetalloproteinases (MMP), such as collagenases, for example of groups I,II, III, IV or V; stromelysin 1, stromelysin 2 or stromelysin 3;metrilysins; gelatinases, such as gelatinase A (MMP 2), andprogelatinase B (MMP 9) and progelatinase A [Pappot et al., Lung Cancer12, 1 (1995), Schmitt et al., Fibronolysis 614, 3 (1992), Monsky et al.,Cancer Biol. 4, 251 (1993), Rochefort et al., Medicine/Sciences 7, 30(1991), Kao et al., 46, 1349 (1986), Fridman et al., Cancer Res. 55,2548 (1995), Ray et al., Eur. Respir. J. 7, 2062 (1994), Cottam et al.,Int. J. Oncol. 2, 861 (1993), Tryggvason et al., Breast Cancer Res. andTreatm. 24, 209 (1993)]; tumor cell surface proteases [surface-expressedproteases=seprase; Monsky et al., Cancer Res. 54, 5702 (1994)]; elastase[Kao et al., Cancer Res. 46, 1355 (1986)]; prostate-specific antigen[Lundwall, Biochem. Biophys. Res. Commun. 161, 1151 (1989), Riegman etal., Biochem. Biophys. Res. Commun. 159, 95 (1989)] or pancreatictrypsinogens [Miszuk-Jamska et al., FEBS Lett. 294, 175 (1991)].

[0055] In accordance with another embodiment of the present invention,the nucleotide sequence for component b) can be extended by the additionof a component b′). This component b′) encodes a ligand (part structureB′) which can bind the active compound to a target structure. Componentb′) is, for example, arranged as shown in FIG. 2. Expression of thenucleic acid construct corresponding to FIG. 2 results in a protein,i.e. B′BCD, which binds to a target structure by way of the ligand (partstructure B′). The part structure C is then cleaved by an enzyme, forexample, cellular proteases, thereby releasing the active compound, i.e.protein B′B.

[0056] In a particular embodiment, said protein B and the part structureD are parts of the natural precursors of protein active compounds, withthe natural cleavage sequence, which connects the part structures B andD, having been replaced by the part structure C; in particular, saidpart structure D is the part structure of a natural precursor of aprotein active compound.

[0057] The novel nucleic acid constructs are preferably composed of DNA.The term “nucleic acid constructs” is understood to mean artificialnucleic acid structures which can be transcribed in the target cells.They are preferably inserted into a vector, with plasmid vectors orviral vectors being particularly preferred.

[0058] Depending on the choice of the promoter element [component a)],the novel nucleic acid constructs express a structural gene [componentsb)+c)+d) or b′)+b)+c)+d)] either nonspecifically, cell-specifically,virus-specifically, under particular metabolic conditions, cellcycle-specifically or in the presence of tetracycline. At least twoidentical or different promoter elements can also be combined togetherfor the purpose of modifying the expression of the structural genedepending on the choice of these promoter elements. Component a) ispreferably activated in endothelial cells, in cells adjoining activatedendothelial cells, in muscle cells, in leukemia cells, in tumor cells,in glia cells, in lymphocytes, in macrophages and/or in synovial cells.

[0059] The part structure B (protein B) of the protein encoded by thenovel structural gene constitutes the actual novel active compound whichis released or activated by cleavage of the part structure C and therebyconverted from the inhibited state, e.g. as protein BCD or as proteinB′BCD, into the active state, e.g. as protein B or as protein B′B.

[0060] According to the invention, this active compound can be an enzymewhich activates or inhibits a biological activation cascade and/or is anactive component of this cascade. Examples of biological activationcascades of this nature are the coagulation system, which can beactivated or inhibited, fibrinolysis, which is preferably activated, thecomplement system, which is likewise preferably activated, or the kininsystem, which is also preferably activated. The active compound can alsobe an enzyme which converts the inactive precursor of a pharmacologicalsubstance into the active substance or which itself is apharmacologically active substance. Particular preference is given to anactive compound (protein B) which is a coagulation factor which isselected from thrombin, factor Va, factor VIIa, factor IXa, factor Xa,TF coagulation-active fragments or factor XIIa; thrombin which ismutated in the region of the Arg-Thr cleavage site (amino acid position327/328); a fibrinolytic protein which is selected from urokinase, tPAor functional hybrids thereof; a complement factor which is selectedfrom CVF, C3b or functional cleavage products thereof; an antithromboticprotein which is selected from protein C, C-1S inhibitor,α1-antitrypsin, hirudin, AT-III, TFPI, PAI-1, PAI-2 or PAI-3; akallikrein; a cytostatic, cytotoxic or inflammation-eliciting protein;an antiangiogenic protein; an immunomodulatory protein; anantiinflammatory protein; a protein which relieves damage to the nervoussystem; a protein which inhibits or neutralizes the neurotoxic effect ofTNFα; an angiogenesis-stimulating protein; a hypotensive protein; anantiviral protein; a cytokine; an interferon; a tumor necrosis factor;oncostatin M or LIF; a cytokine receptor; the moiety of a cytokinereceptor which is external to the cell; a cytokine antagonist; a growthfactor; a growth factor receptor; the moiety of a growth factor receptorwhich is external to the cell; a chemokine; angiostatin; platelet factor4; TIMP 1, TIMP 2 or TIMP 3; a nitroreductase; a β-glucuronidase; acarboxypeptidase; a β-lactamase; a cytosine deaminase; a catalase; aperoxidase; a phosphatase; an oxidase; kallikrein or an endothelial cellnitric oxide synthase.

[0061] The part structure B′ of the protein encoded by the novelstructural gene constitutes the novel ligand for binding the activecompound (protein B) to a target structure. A preferred target structureis the surface of cells, preferably a cell membrane receptor, a cellmembrane antigen, a cell membrane-located adhesion molecule, or theextracellular matrix, for example of endothelial cells, in particular ofactivated or proliferating endothelial cells, tumor cells, muscle cells,in particular smooth muscle cells, fibroblasts, macrophages,lymphocytes, liver cells, kidney cells, synovial cells, inflammatorycells, virus-infected cells, bronchial epithelial cells, glia cells,leukemia cells or cells of other tissues and organs. A particularlypreferred target structure is the surface of activated and/orproliferating endothelial cells.

[0062] Another preferred target structure is constituted by componentsof the extracellular matrix, for example collagens [Prockop et al.,Annu. Rev. Biochem. 64, 403 (1995), Wetzels et al., Am. J. Pathol. 139,451 (1991)]; ficolin [Ichijo et al., J. Biol. Chem. 268, 14505 (1993)];sialoprotein [Bellahcene et al., Cancer Res. 54, 2823 (1994)]; laminin[von der Mark et al., Biochem. Biophys. Acta 823, 147 (1985); Hunt.Expl. Cell Biol. 57, 165 (1989)]; proteoglycans [Schmidtchen et al.,Biomed. Chromatography 7, 48 (1993)] or tenascin [Oyama et al., CancerRes. 51, 4876 (1991); Herlyn et al., Cancer Res. 51, 4853 (1991)].

[0063] The novel ligand (part structure B′) can, for example, be anantibody or an antibody fragment, such as the epitope-binding moiety ofan antibody, Fab, Fv, single-chain Fv or Fc, which binds specifically toa cell membrane antigen or to an antigen on the extracellular matrix, oranother peptide or protein which binds to a receptor on the relevantcell membrane. These include, for example, growth factors, cytokines,interferons, tumor necrosis factor, chemokines, their receptor-bindingpart sequences, peptide hormones, angiotensin, kinin or folic acid. Theligand can also be an adhesion molecule or its adhesion sequence whichbinds to a corresponding molecule on the cell membrane or on theextracellular matrix, or the target cell-binding moiety, anextracellular moiety of an Fc receptor, a target cell-bindingglycoprotein of a virus which has a tropism for selected cells, or apart sequence of the glycoprotein which binds to these cells, or apeptide by whose help the active compound is anchored in the cellmembrane of the cell which is expressing it. Examples of these anchoringpeptides are the transmembrane domains of receptors or virus proteins orglycophospholipid anchors.

[0064] Component d) encodes a peptide (part structure D) which is bondedto protein B or protein B′B by way of part structure C and therebyinhibits the activity of protein B. Component d) can be any arbitrarynucleic acid sequence. Preferably, however, it is composed of nucleicacid sequences which encode endogenous peptides or proteins, in order toavoid or decrease the danger of an immune reaction. In another preferredembodiment, components b) and d) of the novel structural gene encodeendogenous proteins or peptides.

[0065] A considerable number of protein active compounds occur in naturein the form of inactive precursors (protein BSD). A precursor of thistype is activated by enzymes cleaving this precursor into a partstructure which constitutes the actual protein active compound (proteinB) and into an inactive part structure (part structure D). Thisprecursor is cleaved at least one defined amino acid sequence, i.e. theso-called cleavage sequence (part structure S).

[0066] It is a particular part of the subject-matter of this inventionthat this cleavage sequence (part structure S) which occurs naturally inprecursors of protein active compounds is replaced by the part structureC. This replacement is effected by the sequence encoding the partstructure S being replaced by component c), encoding part structure C,in the nucleic acid sequence which encodes the natural precursor(protein BSD). After components a) and, where appropriate, b′) have beenadded on, a novel nucleic acid construct is produced which comprises,for example, components a)b′)b)c)d) or a)b)c)d), the part structure C ofwhose expression product, i.e. protein B′BCD or BCD, respectively, iscleaved by proteases which are formed in tumors or secreted by tumorcells or inflammatory cells, such that the active compound, i.e. proteinB′B or B, can be formed.

[0067] In another embodiment, the novel construct comprises at least twoidentical or different components b)c)d) and/or b′)b)c)d), whichcomponents are linked to each other by way of a so-called internalribosomal entry site (IRES).

[0068] Having been inserted into a nonviral vector or viral vector, thenovel nucleic acid construct is generally, for the prophylaxis and/ortherapy of disorders, administered locally or injected into the bloodcirculation. These disorders particularly include tumor diseases andinflammations. Such inflammations can be triggered, for example, byphysicochemical damage, by an infection or by an immune reaction againstendogenous or foreign tissue.

[0069] The present invention furthermore relates, therefore, to the useof a novel nucleic acid construct for preparing a drug for local orsystemic administration for the prophylaxis and/or therapy of tumors,leukemias, allergies, autoimmune diseases, infections, inflammations,transplant rejection reactions, thromboses, blood vessel occlusions,blood coagulation and blood circulation disturbances, and injuries totissues and/or damage to the nervous system.

[0070] The choice of the components of the novel nucleic acid constructdepends on the disease which is to be treated by administering thenucleic acid construct and can be made as follows:

[0071] Promoter Sequences [component a)]:

[0072] According to the present invention, particular preference isgiven, on the one hand, to promoter sequences [component a)] which arepromoters and activator sequences which can be activated in anunrestricted manner, such as the promoter of RNA polymerase III, thepromoter of RNA polymerase II, etc., the CMV promoter and CMV enhancer,or the SV40 promoter, and, on the other hand, to viral promoter andactivator sequences, such as HBV, HCV, HSV, HPV, EBV, HTLV or HIV.

[0073] For example, in the case of the HIV promoter, the entire LTRsequence, including the TAR sequence [positions ≦−453 to ≧−80, Rosen etal., Cell 41, 813 (1985)] can be used as a virus-specific promoter.

[0074] Metabolically activatable promoter and enhancer sequences, suchas the hypoxia-inducible enhancer, promoters which can be activated in acell cycle-specific manner, such as the promoters of the cdc25C gene,the cyclin A gene, the cdc2 gene, the Bmyb gene, the DHFR gene or theE2F-1 gene, or tetracyline-activatable promoters, such as thetetracycline operator in combination with an appropriate repressor, arealso particularly preferred as component a).

[0075] According to the present invention, nucleotide sequences which,after binding transcription factors, activate the transcription of astructural gene which adjoins them at the 3′ end are also to be used aspromoter sequences.

[0076] In addition, promoters which can be activated in a cell-specificmanner are particularly preferred as component a). These promoterspreferably include promoters or activator sequences composed ofpromoters or enhancers from those genes which preferably encode proteinsin selected cells. For example, promoters for the following proteins arepreferably to be used in the following cells:

[0077] Promoter and activator sequences which are activated inendothelial cells, such as brain-specific, endothelial glucose-Itransporter, endoglin, VEGF receptor 2 (flt-1), VEGF receptor 2 (flk-1,KDR), tiel-1 or tiel-2, B61 receptor (Eck receptor), B61, endothelin,especially endothelin B and endothelin 1, endothelin receptors, inparticular the endothelin B receptor, mannose 6-phosphate receptors, vonWillebrand factor, IL-1α, IL-1β, IL-1 receptor, vascular cell adhesionmolecule (VCAM 1) or synthetic activator sequences.

[0078] As an alternative to natural, endothelial cell-specificpromoters, use can also be made of synthetic activator sequences whichare composed of oligomerized binding sites for transcription factorswhich are preferentially or selectively active in endothelial cells. Anexample is transcription factor GATA 2, whose binding site in theendothelin 1 gene is 5′-TTATCT-3′ [Lee et al., Biol. Chem. 266, 16188(1991), Dormann et al., J. Biol. Chem. 267, 1279 (1992) and Wilson etal., Mol. Cell. Biol. 10, 4854 (1990)].

[0079] Promoters or activator sequences which are activated in cells inthe vicinity of activated endothelial cells, in particular in smoothmuscle cells, are present, for example, in the VEGF gene. Thegene-regulatory sequences for the VEGF gene are the 5′-flanking region,the 3′-flanking region, the c-Src gene or the v-Src gene.

[0080] Steroid hormone receptors and their promoter elements, inparticular the mouse mammary tumor virus promoter, or promoter elementsof the gene encoding tropomyosin, α-actin, α-myosin, the receptor forPDGF, the receptor for FGF, MRF-4, phosphofructokinase A,phosphoglycerate mutase, troponin C, myogens, receptors for endothelinA, desmin or separate “artificial” promoters, are also suitable.Promoter elements to which the factors of the helix-loop-helix (HLH)family (MyoD, Myf 5, myogens and MRF4 [review in Olson and Klein, GenesDev. 8, 1 (1994)]) can bind, as muscle-specific transcription factors,are likewise suitable. The muscle-specific transcription factors alsoinclude the zinc finger protein GATA-4 (Arceci et al., Mol. Cell Biol.13, 2235 (1993), Ip et al., Mol. Cell Biol. 14, 7517 (1994)] and thegroups of the MEF transcription factors [Yu et al., Gene Dev. 6, 1783(1992)].

[0081] The HLH proteins, and also GATA 4, exhibit a similarmuscle-specific transcription not only with promoters frommuscle-specific genes but also in a heterologous context, that is with“artificial” promoters. Examples of such artificial promoters aremultiple copies of the (DNA) binding site for muscle-specific HLHproteins, such as the E box (myo D), e.g. 4× AGCAGGTGTTGGGAGGC,[Weintraub et al., PNAS 87, 5623 (1990)] or multiple copies of the DNAbinding site for GATA 4 of the α-myosin heavy chain gene, e.g.5′-GGCCGATGGGCAGATAGAGGGGGCCGATGGGCAGATAGAGG3′ [Molkentin et al., Mol.Cell Biol. 14, 4947 (1994)].

[0082] Examples of promoters and activator sequences which are activatedin leukemia cells are promoters for c-myc, HSP-70, bcl-1/cyclin D-1,bcl-2, IL-6, IL-10, TNFα, TNFβ, HOX-11, BCR-Ab1, E2A-PBX-1 or PML-RATA.

[0083] Examples of promoters or activator sequences which are activatedin tumor cells are promoter or activator sequences which interact withthe transcription factors which are formed, or are active, in tumorcells. These preferred promoter or activator sequences includegene-regulatory sequences or elements from genes which encode proteinswhich are formed, in particular, in cancer cells or sarcoma cells. Thus,for example, the promoter of the N-CAM protein is used in the case ofsmall-cell bronchial carcinomas, the promoter of the hepatitis growthfactor receptor or of L-plastin is used in the case of ovariancarcinomas, and the promoter of L-plastin or of polymorphic epithelialmucin (PEM) is used in the cas of pancreatic carcinomas.

[0084] Promoters and activator sequences which are activated in gliacells are, in particular, the gene-regulatory sequences or elements fromgenes which encode, for example, the following proteins: the Schwanncell-specific protein periaxin, glutamine synthetase, glia cell-specificprotein (glial fibrillary acid protein=GFAP), the glia cell proteinSlOOb, IL-6 (CNTF), 5-HT receptors, TNFα, IL-10, insulin-like growthfactor receptor I and II or VEGF. The gene-regulatory sequences for theVEGF gene have already been listed above.

[0085] Examples of promoters and activator sequences which are activatedin lymphocytes and/or macrophages are the promoter and activatorsequences of the gene encoding cytokines, cytokine receptors andadhesion molecules, and receptors for the Fc fragment of antibodies.Examples of these are: IL-1 receptor, IL-1α, IL-1β, IL-2, IL-2 receptor,IL-3, IL-3 receptor (α subunit), IL-3 receptor (β subunit), IL-4, IL-4receptor, IL-5, IL-6, IL-6 receptor, interferon regulatory factor 1(IRF-1), (the promoter of IRF-1 is activated to the same extent by IL-6as by IFNγ or IFNβ), IFNγ-responsive promoter, IL-7, IL-8, IL-10, IL-11,IFNγ, GM-CSF, GM-CSF receptor (α chain), IL-13, LIF, macrophage colonystimulating factor (M-CSF) receptor, type I and II macrophage scavengerreceptors, MAC-1 (leukocyte function antigen), LFA-1α (leukocytefunction antigen) or p150,95 (leukocyte function antigen).

[0086] Examples of promoter and activator sequences which are activatedin synovial cells are the promoter sequences for matrixmetalloproteinases (MMP), for example for: MMP-1 (interstitialcollagenase), or MMP-3 (stromelysin/transin). These also include thepromoter sequences for tissue inhibitors of metalloproteinases (TIMP),for example TIMP-1, TIMP-2 and TIMP-3.

[0087] According to the present invention, several of the promotersequences which have been listed by way of example can be combined witheach other in order to achieve the highest possible target cellspecificity in the expression of the novel nucleic acid construct. Twoidentical promoters can also be combined. Several promoter sequences canbe combined, for example, using chimeric promoters or hybrid promoters.A chimeric promoter is the combination of an upstream activatorsequence, which can be activated cell-specifically, metabolically orvirus-specifically, with a downstream promoter module which binds thetranscription factors of the CDF and CHF families or the E2F and CHFfamilies and can thereby inhibit activation of the upstream activatorsequence in the GO and Gi phases of the cell cycle (Lucibello et al.,EMBO J. 14, 132 (1994)].

[0088] In the case of hybrid promoters, the TATA box of a promoter is,for example, mutated, with this mutation being compensated for by acorresponding mutation in the gene for a TATA-binding protein, and thisTATA-binding protein being under the control of another promoter.

[0089] Nucleic Acid Sequence Component b′)1. Which Encodes a Ligand(Part Structure B′):

[0090] According to the present invention, the ligand is a substancewhich binds a membrane antigen to a receptor or to an adhesion moleculeon the target cell or which is integrated in the cell membrane and/orbinds to the extracellular matrix. Reviews of the important cytokinesand growth factors and their receptors, adhesion molecules andextracellular matrix proteins are provided by Ayad et al., TheExtracellular Matrix, Academic Press 1994; Callard et al., The Cytokine,Academic Press 1994; Pigott et al., The Adhesion Molecule, AcademicPress 1994, and Barclay et al., The Leucocyte Antigen, Academic Press1994.

[0091] Examples of substances which bind to receptors are growthfactors, such as VEGF, PDGF, EGF, TGFA, TGFβ, KGP, SDGF, FGF, IGF, HGF,NGF, BDNF, neurotrophins, BMF, bombesin, M-CSF, thrombopoietin,erythropoietin, SCF, SDGF, oncostatin, PDEGF or endothelin-1, cytokines,such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, interferons α, β and γ, tumornecrosis factors TNFα and TNFβ, chemokines, such as RANTES, MCAF, MIP-1αor MIP-1β, NAP or β-thromboglobulin, peptide hormones, such as SRH, SIHor STH, MRH or MSH, PRH, PIH or prolactin, LH-RH, FSH-RH, LH/ICSH orFSH, TRH or TSH, CRH or ACTH, angiotensin, kinins, homologs or analogsthereof, or vitamins, such as folic acid.

[0092] According to the present invention, the ligand can also be anadhesion molecule, a part of an adhesion molecule or an analog of anadhesion molecule which binds to a corresponding adhesion molecule whichis located in the cell membrane or to another specific binding structurefor an adhesion molecule on the target cell or in the extracellularmatrix.

[0093] Examples of such adhesion molecules which are capable offunctioning as ligands are Lewis X (for GMP-140), S Lewis X (forELAM-1), LFA-1 (for ICAM-1 and ICAM-2), MAC-1 (for ICAM-1), VLA-4 (forVCAM-1), PECAM (for PECAM), vitronectin (for the vitronectin receptor),GMP-140 (for Lewis X), S Lewis X (for ELAM-1), ICAM-1, ICAM-2 (for LFA-1and MAC-1), VCAM-1 (for VLA-4), fibronectin (for VLA-4), laminin (forVLA-6), laminin (for VLA-1, VLA-2 and VLA-3), fibrinogen (forGPIIb-IIIa), B7 (for CD28), CD28 (for B7), CD40 (for CD40L) or CD40L(for CD40).

[0094] According to the present invention, the ligand can also be theextracellular moiety of an Fc receptor [Dougherty et al., TransfusionScience 17, 121 (1996)]. Furthermore, the ligand can also be an antibodymolecule or the epitope-binding moiety of an antibody molecule. Themurine monoclonal antibodies should preferably be employed in humanizedform. The humanization is effected in the manner described by Winter etal. Nature 349, 293 (1991) and Hoogenbooms et al. Rev. Tr. Transfus.Hemobiol. 36, 19 (1993).

[0095] Recombinant antibody fragments are either prepared directly fromexisting hybridomas or are isolated from libraries of murine or humanantibody fragments [Winter et al., Annu. Rev. Immunol. 12, 433 (1994)]using the phage-display technique [Smith, Science 228, 1315 (1985)]. Theantibody fragments are then employed directly, at the genetic level, forfurther manipulations, e.g. for fusion with other proteins.

[0096] In order to prepare recombinant antibody fragments fromhybridomas, the genetic information which encodes the antigen-bindingdomains (VH and VL) of the antibodies is obtained by isolating the mRNA,reverse-transcribing the RNA into cDNA and then amplifying the cDNA bymeans of the polymerase chain reaction [Saiki et al., Science 230, 1350(1985)] and using oligonucleotides which are complementary to the 5′ and3′ ends of the variable fragments (Orlandi et al., 1989). The VH and VLfragments are then cloned into bacterial expression vectors, for examplein the form of Fv fragments [Skerra & Pluckthun, Science 240, 1038(1988)], single-chain Fv fragments (scFv) [Bird et al., Science 242, 423(1988), Huston et al., PNAS-USA 85, 5879 (1988)] or as Fab fragments[Better et al., Science 240, 1041 (1988)].

[0097] The phage-display technique can also be used to isolate newantibody fragments directly from antibody libraries (immune libraries ornaive libraries) of murine or human origin. In the phage-display ofantibody fragments, the antigen-binding domains are cloned, as proteinfusions with the coat protein g3P of filamentous bacteriophages, eitherinto the phage genome [McCafferty et al., Nature 348, 552 (1990)] orinto phagemid vectors [Breitling et al., Gene 104, 147 (1991)] in theform of scFv fragments [McCafferty et al., Nature 348, 552 (1990)] or asFab fragments [Hoogenboom et al., Nucl. Acid Res. 19, 4133 (1991),Barbas et al., PNAS-USA 88, 7978 (1991)]. Antigen-binding phages areselected on antigen-loaded plastic vessels (panning) [Marks et al., J.Mol. Biol. 222, 581 (1991)], on antigen-conjugated, paramagnetic beads[Hawkins et al., J. Mol. Biol. 226, 889 (1992)] or by binding to cellsurfaces [Marks et al., Bio/Technol. 11, 1145 (1993)].

[0098] Immune libraries are prepared by subjecting the variable antibodyfragments from the B lymphocytes of immunized animals [Sastry et al.,PNAS-USA 86, 5728 (1989), Ward et al., Nature 341, 544 (1989), Clacksonet al., Nature 352, 624 (1991)] or patients [Mullinax et al., PNAS-USA,87, 8095 (1990), Barbas et al., PNAS-USA, 88, 7978 (1991)] to PCRamplification. For this, use is made of combinations of oligonucleotideswhich are specific for murine [Orlandi et al., PNAS-USA, 86, 3833(1989), Sastry et al., PNAS-USA, 86, 5728 (1989)] or humanimmunoglobulin genes [Larrick et al., BBRC 160, 1250 (1989)] or for thehuman immunoglobulin gene families [Marks et al., Eur. J. Immunol. 21,985 (1991)].

[0099] Naive libraries can be prepared, for example, using nonimmunizeddonors as the source of the immunoglobulin genes [Marks et al., J. Mol.Biol. 222, 581 (1991)]. Alternatively, immunoglobulin germ line genescan be used to prepare semisynthetic antibody repertoires, with thecomplementarity-determining region 3 of the variable fragments beingamplified by PCR using degenerate primers [Hoogenboom & Winter, J. Mol.biol. 227, 381 (1992), Barbas et al., PNAS-USA, 89, 4457 (1992), Nissimet al., EMBO J. 13, 692 (1994), Griffiths et al., EMBO J. 13, 3245(1994)]. As compared with immune libraries, these so-called single-potlibraries have the advantage that antibody fragments against a largenumber of antigens can be isolated from one single library [Nissim etal., EMBO J, 13, 692 (1994)].

[0100] The phage-display technique can be used to increase the affinityof antibody fragments still further, with new libraries being preparedfrom already existing antibody fragments by random [Hawkins et al., J.Mol. Biol. 226, 889 (1992), Gram et al., PNAS-USA, 89, 3576 (1992)],codon-based [Glaser et al., J. Immunol. 149, 3903 (1992)] orsite-directed mutagenesis [Balint & Larrick, Gene 137, 109 (1993)], byshuffling the chains of individual domains with those of fragments fromnaive repertoires [Marks et al., Bio/Technol 10, 779 (1992)] or by usingbacterial mutator strains [Low et al., J. Mol. Biol. 26, 359 (1996)],and antibody fragments having improved properties being isolated byreselecting under stringent conditions [Hawkins et al., J. Mol. Biol.226, 889 (1992)]. In addition, murine antibody fragments can behumanized by a stepwise replacement of one of the variable domains witha human repertoire and then selecting with the original antigen (guidedselection) [Jespers et al., Bio/Technol, 12, 889 (1994)]. Alternatively,murine antibodies are humanized by specifically replacing thehypervariable regions of human antibodies with the corresponding regionsof the original murine antibody [Jones et al., Nature 321, 522 (1987)].

[0101] According to the present invention, the ligand can also be thenucleotide sequence encoding a coat protein, or a part of a coatprotein, of viruses which specifically bind to selected cells by way oftheir coat protein.

[0102] The ligand can also be a peptide, with whose help the activecompound (protein B) is anchored in the cell membrane of the expressingcells. These anchoring peptides include the transmembrane domains ofcell membrane-located receptors or of virus proteins, such as thetransmembrane sequence of human macrophage colony-stimulating factor[DNA position ≦1485 to ≧1554; Cosman et al., Behring Inst. Mitt. 83, 15(1988)] or the DNA sequence for the signal and transmembrane regions ofhuman respiratory syncytial virus (RSV) glycoprotein G [amino acids 1 to63 or their part sequences, amino acids 38 to 63; Vijaya et al., Mol.Cell Biol. 8, 1709 (1988); Lichtenstein et al., J. Gen. Virol. 77, 109(1996)] or the DNA sequence for the signal and transmembrane region ofinfluenza virus neuraminidase [amino acids 7 to 35 or the part sequenceof amino acids 7 to 27, Brown et al., J. Virol. 62; 3824 (1988)].

[0103] However, the nucleotide sequence for a glycophospholipid anchor[review of glycophospholipid-anchored membrane proteins in Ferguson etal., (Arn. Rev. Biochem. 57, 285 (1988))] can also be inserted for thepurpose of anchoring the active compound in the cell membrane of thetransduced cells which form the active compound. Glycophospholipidanchors have been described, for example, for CEA [DNA position <893to >1079; Berling et al., Cancer Res. 50 6534 (1990)], for N-CAM[Cunningham et al., Science 236, 799 (1987)] and for other membraneproteins such as Thy-1 [Clissold, Biochem. J. 281, 129 (1992)] or CD16[Selvaray et al., Nature 333, 565 (1988)].

[0104] The choice of the ligand depends, first and foremost, on thetarget cell which is to be transduced with the nucleic acid construct.Ligands for activated endothelial cells are examples of this. Within themeaning of the invention, these ligands include antibodies or antibodyfragments which are directed against membrane structures of endothelialcells, as have been described, for example, by Burrows et al. Pharmac.Ther. 64, 155 (1994), Hughes et al., Cancer Res. 49, 6214 (1989) andMaruyama et al., PNAS-USA 87, 5744 (1990). In particular, theseantibodies include antibodies against actin, angiotensin II receptors,antibodies against receptors for growth factors such as VEGF, FGF, PDGFor EGF, and antibodies against adhesion molecules, for example againstthe vitronectin receptor or ICAM 3.

[0105] The ligands furthermore include all active compounds which bindto membrane structures or membrane receptors on endothelial cells.Examples of these are IL-1 or growth factors, or their fragments or partsequences thereof, which bind to receptors which are expressed inendothelial cells, for example PDGF, bFGF, VEGF or TGFβ [Pusztain etal., J. Pathol. 169, 191 (1993)].

[0106] The ligands furthermore include adhesion molecules which bind toactivated and/or proliferating endothelial cells. Adhesion molecules ofthis nature, such as Slex, LFA-1, MAC-1, LECAM-1, VLA-4 or vitronectin,have already been described [Augustin-Voss et al., J. Cell Biol. 119,483 (1992), Pauli et al., Cancer Metast. Rev. 9, 175 (1990), Honn etal., Cancer Metast. Rev. 11, 353 (1992), Pigott et al., The AdhesionMolecule, Academic Press (1994)].

[0107] The ligands within the meaning of this invention also include, inparticular, glycoproteins from the coats of viruses which have a tropismfor endothelial cells. Examples of these viruses are filoviruses, suchas Marburg virus with its coat proteins GP (glycoprotein) and sGP(second glycoprotein) or Ebola virus, in each case with its coatproteins GP and sG, cytomegalovirus, particularly with its gB protein,herpes simplex virus type I, HIV-1 virus, measles virus, Hantaan virus,alphaviruses, such as Semliki forest virus, epidemic hemorrhagic fevervirus, polio virus or enteroviruses, such as ECHO 9, ECHO 12 andCoxsackie B3.

[0108] Antibodies or antibody fragments which are directed againstmembrane structures of muscle cells, in particular of smooth musclecells, are examples of ligands for muscle cells. Examples of antibodiesof this nature are antibody 10F3, antibodies against actin, antibodiesagainst angiotensin II receptors, antibodies against receptors forgrowth factors or antibodies, for example, against EGF receptors,against PDGF receptors or against FGF receptors, or antibodies againstendothelin A receptors.

[0109] The ligands furthermore include nucleotide sequences for activesubstances which bind to membrane structures or membrane receptors onmuscle cells [Pusztai et al., J. Pathol. 169, 191 (1993), Harris, Curr.opin. Biotechnol. 2, 260 (1991)]. Examples of these ligands are growthfactors, or their fragments or part sequences thereof, which bind toreceptors which are expressed in smooth muscle cells, for example PDGF,EGF, TGFβ, TGFα, FGF or endothelin A.

[0110] The ligands also include glycoproteins from the coats of thoseviruses which have a tropism for muscle cells. An example of theseviruses is cytomegalovirus [Speir et al., Science 265, 391 (1994)].

[0111] Examples of ligands for activated macrophages and/or activatedlymphocytes are, in addition, nucleotide sequences which encodesubstances which bind specifically to the surface of immune cells. Thesesubstances include antibodies or antibody fragments which are directedagainst membrane structures of immune cells, as have been described, forexample, by Powelson et al., Biotech. Adv. 11, 725 (1993) and Barclay etal., The Leucocyte Antigen, Academic Press (1994). The ligands alsoinclude monoclonal or polyclonal antibodies or antibody fragments whichbind, by their antigen-binding variable moiety, to Fcγ, Fcε or Fcμreceptors of immune cells [Rojanasakul et al., Pharm. Res. 11, 1731(1994)]. They furthermore include the Fc fragment of human monoclonal orpolyclonal immunoglobulin.

[0112] The ligands furthermore include all substances which bind tomembrane receptors on the surface of immune cells. These substancesinclude cytokines, such as IL-1, IL-2, IL-3, IL-4, IL-6, IL-10, TNFα,GM-CSF and M-CSF, and also growth factors, such as EGF, TGF, FGF, IGF orPDGF, or their fragments or part sequences thereof, which bind toreceptors which are expressed in immune cells [Callard et al., TheCytokine, Academic Press (1994)]. The ligands also include adhesionmolecules and other ligands which bind to cell membrane structures onmacrophages, and in spleen, liver, lung and other tissues [Pigott etal., The Adhesion Molecule, Academic Press (1994), Perales et al., Eur.J. Biochem. 226, 255 (1994)].

[0113] The ligands within the meaning of this invention also includeglycoproteins from the coats of those viruses which have a tropism forlymphocytes and/or macrophages. Examples of these macrophage-infectingviruses are HIV-1, in particular those strains having mutations in theV3 region of gp120 which result in increased binding to macrophages,HIV-2, Hantaviruses, for example Punmalavirus, cytomegalovirus,respiratory syncytial virus, herpes simplex virus or filoviruses.

[0114] Examples of lymphocyte-infecting viruses are varicella zostervirus (VZV), since VZV infects T cells in particular, herpesvirus 6 (HHV6), since HHV 6 likewise infects T cells in particular, rabies virus,since rabies virus coat protein binds to TH2 cells in particular, HIV-1,since glycoprotein gp120 binds preferably to the CD4 molecule of Tcells, HTLV-II, since HTLV-II infects B cells in particular, HTLV-I,since HTLV-I infects T cells in particular, influenza C viruses, sinceinfluenza C viruses bind to N-acetyl-9-β-acetylneuraminic acid (Neu 5,9Ac), which preferentially occurs on B lymphocytes and to a lesserextent, or not at all, on T lymphocytes, by way of thehemagglutinin-esterase fusion (HEF) protein, influenza C viruses havinga mutation in nucleotide position 872, which encodes position 284 of theamino acid sequence of the HEF, for example with the threonine beingreplaced with isoleucine, since the surface protein HEF which possessesthis mutation has a markedly stronger affinity for theN-acetyl-9-O-acetylneuraminic acid receptor than does the wild-typevirus, cleavage products of the influenza C virus HEF which contain thestructure for binding to N-acetyl-9-β-acetylneuraminic acid. Thisbinding structure is defined by the catalytic triad serine 71, histidine368 or 369 and aspartic acid 261, Epstein-Barr virus, since EBV infectsB cells in particular, herpes simplex virus 2, since HSV-2 infects Tcells in particular, or measles virus.

[0115] Examples of ligands for synovial cells and inflammatory cellswhich are to be mentioned are nucleic acid sequences which encodeantibodies or antibody fragments which bind, by their variable domains,to membrane structures of synovial cells or inflammatory cells. Examplesof these membrane structures are vimentin [Miettinen et al., Am. J.Pathol. 117, 18 (1984)], fibronectin [Wojciak et al., Clin. Exp.Immunol. 93, 108 (1993)] or Fc receptors. These ligands also includeantibodies or antibody fragments which bind to the Fc receptor by theirconstant domains [Rojanasakul et al., Pharm. Res. 11, 1731 (1994)].

[0116] These ligands furthermore include all active compounds which bindto membrane structures or membrane receptors on synovial cells. Examplesof these are cytokines or growth factors, or their fragments or partsequences thereof, which bind to receptors which are expressed bysynovial cells, for example IL-1-RA, TNFα, IL-4, IL-6, IL-10, IGF orTGFβ [Callard et al., The Cytokine, Academic Press (1994)].

[0117] Examples of ligands for virus-infected cells which are to bementioned are nucleic acid constructs which encode antibodies orantibody fragments which are directed against the viral antigens whichare located on the cell membrane of virus-infected cells. Antibodies ofthis nature are directed, for example, against antigens of HBV, HCV,HSV, HPV, HIV, EBV or HTLV.

[0118] Examples of ligands for liver cells and other tissue cells areall substances which bind to membrane structures or membrane receptorson the surface of liver cells. Examples of these are growth factors,such as cytokines, EGF, TGF, FGF or PDGF, or their fragments or partsequences thereof, which bind to receptors which are expressed in cellsof this nature.

[0119] These ligands furthermore include ligands which bind to cellmembrane structures which are selective for particular tissues. Examplesare: Ligand Tissue cells Transferrin receptor Transferrin Liver, othertissue cells Insulin receptor Insulin Liver, other tissue cells Fcγreceptors Immunoglobulin G Reticuloendothelial system, other tissuecells

[0120] These ligands and membrane structures are reviewed in Perales etal., Eur. J. Biochem. 226, 255 (1994).

[0121] The ligands particularly include glycoproteins from the coats ofviruses which have a tropism for selected cells, such as for bronchialepithelial cells (respiratory syncytial virus), liver cells (hepatitis Cvirus), filoviruses, Marburg virus by way of the asialoglycoproteinreceptor of liver cells, hepatitis B virus, with liver cells preferablybinding to the preS2 and presi domains of HBV by way of theasialoglycoprotein receptor, hepatitis D virus, liver-sinusoiddl cells,and heptatis B virus, with HBV being bound by way of fibronectin.

[0122] Examples of ligands for glia cells are nucleic acid sequenceswhich encode antibodies or antibody fragments which are directed againstmembrane structures of glia cells, as have been reported, for example,by Mirsky et al. [Cell and Tissue Res. 240, 723 (1985)], Coakham et al.[Prog. Exp. Tumor Res. 29, 57 (1985)] and McKeever et al. [Neurobiol. 6,119 (1991)]. These membrane structures furthermore include neuraladhesion molecules such as N-CAM, in particular its polypeptide chain C[Nybroe et al., J. Cell Biol. 101, 2310 (1985)]. These ligandsfurthermore include all active compounds which bind to membranestructures or membrane receptors on glia cells. Examples of these activecompounds are insulin and insulin-like growth factor, and thosefragments of these growth factors which bind to the relevant membranereceptors.

[0123] The ligands within the meaning of the invention additionallyinclude nucleic acid sequences which encode glycoproteins of the coatsof those viruses which have a tropism for glia cells.

[0124] These viruses include, for example, HIV-1 subtype JRF1 or herpessimplex virus I.

[0125] Examples of ligands for leukemia cells include nucleic acidconstructs which encode antibodies or antibody fragments which aredirected against membrane structures of leukemia cells. A large numberof monoclonal antibodies of this nature have already been described fordiagnostic and therapeutic procedures [Kristensen, Danish MedicalBulletin 41, 52 (1994); Schranz, Therapia Hungarica 38, 3 (1990);Drexler et al., Leuk. Res. 10, 279 (1986); Naeim, Dis. Markers 7, 1(1989); Stickney et al., Curr. Opin. Oncol. 4, 847 (1992); Drexler etal., Blut 57, 327 (1988); Freedman et al., Cancer Invest. 9, 69 (1991)].Depending on the type of leukemia, monoclonal antibodies, or theirantigen-binding antibody fragments, of the following specificity are,for example, suitable as ligands:

[0126] AML cells having the membrane antigens CD13, CD14, CD15, CD33,CAMAL and sialosyl-Le; B-CLL cells having the membrane antigens CD5,CD1c and CD23, and also idiotypes and isotypes of the membraneimmunoglobulins; T-CLL cells having the membrane antigens CD33, M38,IL-2 receptors and T cell receptors; and ALL cells having the membraneantigens CALLA and CD19, and also non-Hodgkin's lymphoma.

[0127] The ligands furthermore include all active compounds which bindto membrane structures or membrane receptors of leukemia cells. Examplesof these are growth factors, or their fragments or part sequencesthereof, which bind to receptors which are expressed in leukemia cells.

[0128] Growth factors of this nature have already been described[reviews in Cross et al., Cell 64, 271 (1991); Aulitzky et al., Drugs48, 667 (1994); Moore, Clin. Cancer Res. 1, 3 (1995); Van Kooten et al.,Leuk. Lymph. 12, 27 (1993)]. For example, they include IFNα, in the caseof non-Hodgkin's lymphomas, IL-2, particularly in the case of T cellleukemias, FGF in the case of T cell, monocytic, myeloid, erythrocyticand megakaryoblastic leukemias, TGFβ in the case of leukemias, orretinoids, e.g. retinoic acid, in the case of acute promyelocyticleukemia.

[0129] Examples of ligands for tumor cells include nucleic acidsequences which encode antibodies, and fragments of these antibodies,which are directed against membrane structures on tumor cells.Antibodies of this nature have been reviewed, for example, by Sedlaceket al., Contrib. to Oncol. 32, Karger Verlag, Munich (1988) and Contrib.to Oncol. 43, Karger Verlag, Munich (1992).

[0130] Other examples are antibodies against sialyl Lewis, peptides ontumors which are recognized by T cells, proteins expressed by oncogenes,gangliosides such as GD3, GD2, GM2, 9-O-acetyl-GD3 and fucosyl-GM1,blood group antigens and their precursors, antigens on polymorphicepithelial mucine or antigens on heat shock proteins.

[0131] Nucleic Acid Sequence [Component b)] Which Ecodes An ActiveCompound (Protein B):

[0132] The active compound (protein B) according to the presentinvention can be a substance which, for example, intervenes in abiological activation cascade and/or is an active component of thiscascade. These substances include active compounds which activate thecoagulation cascade, for example thrombin [MacGillivray et al., Ann.N.Y. Acad. Sci. 485, 73 (1986)], thrombin which is mutated in the regionof the Arg-Thr cleavage site (amino acid position 327/328), factor Va[Cripe et al., Biochem. 31, 3777 (1992), Jenny et al., PNAS-USA 84, 4846(1987)], factor VIIa [O'Hara et al., PNAS-USA 84, 5158 (1987)], factorIXa [Yoshitake et al., Biochem. 24, 3736 (1985)], factor Xa [Messier etal., Gene 99, 291 (1991)] or tissue factor and coagulation-activefragments thereof [Morrissey et al., Cell 50, 29 (1987); Scarpati etal., Biochem. 26, 5234 (1987); Spicer et al., PNAS-USA 84, 5148 (1987);Rehemtulla et al., Thromb. Heamost. 65, 521 (1991)] or which inhibit thecoagulation cascade or which activate fibrinolysis, for example theplasminogen activator inhibitors PAI-1, PAI-2 and PAI-3, hirudin,protein C, serine proteinase inhibitors, such as C-1S inhibitor,α1-antitrypsin or antithrombin III, tissue factor pathway inhibitor(TFPI), plasminogen activators such as urokinase, tissue plasminogenactivator (tPA), or hybrids thereof, or which activate the complementcascade, for example cobra venom factor (CVF) or part sequences of CVFwhich correspond functionally to human complement factor C3b, i.e. whichare able to bind to complement factor B and which, after having beencleaved by factor D, constitute a C3 convertase (the DNA sequence forCVF and its part sequences were described by Fritzinger et al., Proc.Natl. Acad. Sci. USA 91, 12775 (1994)), human complement factor C3b (theDNA sequence for C3 and its part sequences were published by De Bruijnet al., Proc. Natl. Acad. Sci. USA 82, 708 (1985), cleavage products ofhuman complement factor C3 which resemble CVF functionally andstructurally (such cleavage products have been described by O'Keefe etal., J. Biol. Chem. 263, 12690 (1988) or which activate the kininsystem, the complement system and/or the coagulation system, for exampleactivated Hagemann factor (F XIIa) [Shibuya et al., Biochem, Biophys.Acta 1206, 63 (1994), Que et al., Biochem. 25, 1525 (1986), Tripodi etal., Nucl. Acid Res. 14, 3146 (1986)] or kallikrein [Chen et al.,Biochem. J. 307, 481 (1995), Fukushima et al., Biochem. 24, 8037(1985)].

[0133] The active compound (protein B) can also be a cytostatic,cytotoxic or inflammation-eliciting protein, such as perforin, granzyme,cytokines, such as IL-1, IL-2, TL-4, IL-12, IL-3, IL-5, human leukemiainhibitory factor (LIF), IL-7, IL-11, IL-13, GM-CSF, G-CSFb or M-CSF,interferons, such as IFNα, IFNβ or IFNγ, TNF, such as TNFα or TNFβ,oncostatin M, sphingomyelinase [Jarvis et al., PNAS USA 91, 73 (1994)],magainin and magainin derivatives [Cruciani et al., PNAS USA 88, 3792(1991)]; Jacob et al., Ciba Found. symp. 186, 197 (1994); Peck-Miller etal., Cancer Chemother. Pharmac. 32, 109 (1993)] or chemokines, such asRANTES (MCP-2), monocyte chemotactic and activating factor (MCAF), IL-8,macrophage inflammatory protein 1 (MIP-1α or MIP-1β) or neutrophilactivating protein 2 (NAP-2).

[0134] The active compound (protein B) can also be an antiangiogenicprotein, such as angiostatin, interferons, such as IFNα, IFNβ or IFNγ,platelet factor 4, IL-12, TIMP-1, TIMP-2 or TIMP-3.

[0135] The active compound (protein B) can also be an enzyme which isable to convert an inactive precursor of a pharmacological activesubstance, for example a cytostatic agent, into the active substanceitself. Examples of such active compounds are bacterial nitroreductase,bacterial β-glucuronidase, plant β-glucuronidase derived from Secalecereale, human β-glucuronidase, human carboxypeptidase (CB), e.g. mastcell CB-A or pancreas CB-B, or bacterial carboxypeptidase, bacterialβ-lactamase, bacterial cytosine deaminase, human catalase or peroxidase,phosphatase, in particular human alkaline phosphatase or human acidprostate phosphatase, type 5 acid phosphatase, oxidase, in particularhuman lysyl oxidase or human acid D-aminooxidase, peroxidase, inparticular human glutathione peroxidase, human eosinophilic peroxidaseor human thyroid peroxidase.

[0136] The active compound (protein B) can also be a protein whichaffects the immune system, for example a protein having an antiallergiceffect, such as IFNβ, IFNγ, IL-10, soluble IL-4 receptors, IL-12 orTGFβ, or a protein which can prevent the rejection of transplantedorgans, such as IL-10, TGFβ, soluble IL-1 receptors, soluble IL-2receptors, IL-2 receptor antagonists or soluble IL-6 receptors, or aprotein for the therapy of antibody-mediated autoimmune diseases, forexample TGFβ, IFNα, IFNβ, IFNγ, IL-12, soluble IL-4 receptors or solubleIL-6 receptors, or a protein for the therapy of cell-mediated autoimmunediseases, for example IL-6, IL-9, IL-10, IL-13, TNFα, IL-4 or TNFβ, or aprotein for the therapy of arthritis. According to the presentinvention, structural genes can also be selected whose expressed proteindirectly or indirectly inhibits inflammation, for example in a joint,and/or promotes the reconstitution of extracellular matrix (cartilageand connective tissue) in the joint. These expressed proteins include,for example, IL-1 receptor antagonists (IL-1-RA), since IL-1-RA inhibitsthe binding of IL-1α and IL-1β, soluble IL-1 receptor, since solubleIL-1 receptor binds and inactivates IL-1, IL-6, since IL-6 increasessecretion of TIMP and superoxides and decreases secretion of IL-1 andTNFa by synovial cells and chondrocytes, soluble TNF receptor, sincesoluble TNF receptor binds and activates TNF, IL-4, since IL-4 inhibitsthe formation and secretion of IL-1, TNFα and MMP, IL-10, since IL-10inhibits the formation and secretion of IL-1, TNFα and MMP and increasesthe secretion of TIMP, insulin-like growth factor (IGF-1), since IGF-1stimulates the synthesis of extracellular matrix, TGFβ, especially TGFβ1and TGFβ2, since TGFβ stimulates the synthesis of extracellular matrixsuperoxide dismutase, or TIMP (tissue inhibitors of metalloproteinases),especially TIMP-1, TIMP-2 or TIMP-3.

[0137] The active compound (protein B) can also be a protein forrelieving damage to the nervous system, for example a growth factor,such as FGF, nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4) or ciliaryneurotrophic factor (CNTF), or a cytokine, or a cytokine inhibitor,which is able to inhibit or neutralize the neurotoxic effect of TNFα,for example TGFβ, soluble TNF receptors, IL-10, since IL-10 inhibits theformation of IFNγ, TNFα, IL-2 and IL-4, soluble IL1 receptors, such asIL-1 receptor I or IL-1 receptor II, since soluble IL-1 receptorsneutralize the activity of IL-1, IL-1 receptor antagonist or solubleIL-6 receptors.

[0138] The active compound (protein B) can also be a protein whichstimulates angiogenesis, for example VEGF or FGF.

[0139] The active compound (protein B) can furthermore be a proteinwhich lowers blood pressure, for example kallikrein or endothelial cellnitric oxide synthase.

[0140] The active compound (protein B) can also be a protein for thetherapy of chronic infectious diseases, for example a protein whichexhibits cytostatic or cytotoxic effects, or an enzyme which cleaves aprecursor of an antiviral or cytotoxic substance into the activesubstance, or a cytotoxin having an antiviral effect or a growth factorhaving an antiviral effect. Examples are IFNα, IFNβ, IFNγ, TNFβ, TNFα,IL-1 or TGFβ.

[0141] The present invention furthermore relates to a nucleic acidconstruct in which two identical or two different DNA sequences, whichencode identical or different active compounds (protein B) [component b)and b″)] are combined.

[0142] In order to ensure that both DNA sequences are expressed, thecDNA of an internal ribosome entry site (IRES) is preferablyintercalated, as a regulatory element, between the two structures. Aninternal ribosome entry site makes it possible to express two DNAsequences which are linked to each other by way of an IRES. IRESs ofthis nature have been described, for example, by Montford and Smith TIG11, 179 (1995); Kaufman et al., Nucl. Acids Res. 19, 4485 (1991); Morganet al., Nucl. Acids Res. 20, 1293 (1992); Dirks et al., Gene 128, 247(1993); Pelletier and Sonenberg, Nature 334, 320 (1988) and Sugitomo etal., BioTechn. 12, -694 (1994). Thus, for example, the cDNA for thepolio virus IRES sequence (positions ≦140 to ≧630 of the 5′ UTR[Pelletier and Sonenberg, Nature 334, 320 (1988)] can be used to linkthe DNA of component c) to the DNA of component d).

[0143] Nucleic Acid Sequences [Component c)] Which Encode TheProtease-Cleavable Part Structure C:

[0144] According to the present invention, part stucture C comprises anamino acid sequence which is cleaved by proteases which are formed intumors or by tumor cells or inflammatory cells. The nucleic acidsequence for this part structure C is inserted, for example, into thenucleic acid sequence of the naturally occurring precursor (protein BSD,where S is the naturally occurring cleavage sequence) of the relevantactive compound (protein B) in place of the cleavage sequence S suchthat this recombinant nucleic acid expresses protein BCD or B′BCD.

[0145] The nucleic acid sequence encoding part structure C is chosendepending on the protease which is predominantly secreted in the tumoror in the inflammation.

[0146] The following part structures C may, for example, be employed forthe following enzymes [Barrett et al., Mammalian Proteases, AcademicPress, London (1980), Panchal et al., Nature Biotechnol. 14, 852 (1996);Pigott et al., Ayad et al., The extracellular Matrix, Academic press(1994); Yoshida et al., Int. J. Cancer 63, 863 (1995), Petersen et al.,J. Biol. Chem. 265, 6104 (1990); Cramer et al., J. Urology 156, 526(1995); Forsgen et al., FEBS Lett. 213, 254 (1987) Zhang et al. Chin.Chem. 41, 1567, (1995)]: Part structure C cleavage Enzyme A6 A5 A4 A3 A2A1 A-1 (A-2) Plasminogen Cys Pro Gly Arg Val (Ile) (Val) activator GlnGly Arg Gly Gly Arg Pro Arg Phe Lys Gly Lys Arg Prostrate- Pro Arg PheLys Ile (Ile) (Val) specific Arg Pro Tyr antigen Arg Arg Phe Phe Leu(Ile) (His) (Val) Tyr Ile Val Ser Phe Ser Ile Gln Tyr Ile Val Gly SerGln Gln Leu Leu Ile Val Gly Ile Ser Ser Gln Tyr Ile Val Cathepsins ProArg Phe Lys Ile Ile (Val) Tyr Lys Ser Arg Met (Ile) Lys Met Arg Arg(Ile) Ile Arg Arg Arg (Ile) Arg Ala Arg Leu (Ile) Gln Ala Arg Phe (Ile)Lys Leu Arg Leu (Ile) Lys Arg Val (Ile) Lys Phe Arg Stromelysins Gly GlyGly Ala Gln (Leu) Gln Leu Gly Val Met (Gln) Ala Ala Ala Ser Leu (Lys)Val Ala Val Ser Ala (Lys) Leu Ala Ala Asn Leu (Arg) Collagenase I GlyPro Gln Gly Ile (Ala) Gly Pro Gln Gly Leu (Leu) II Gly Pro Gln Gly Leu(Ala) III Gly Ile Ala Gly Ile (Thr) VIII Gly Leu Pro Gly Ile (Gly) GlyPhe Pro Gly Ile (Gly) XI Gly Pro Ala Gly Ile (Ser) Gly Pro Ala Gly Ile(Ala) Plasminogen Ser Gly Thr Glu Ile (Val)

[0147] Nucleic Acid Sequences [Component d)] Which Encode Part StructureD:

[0148] According to the present invention, the nucleic acid sequence[component d)] encodes a peptide (part structure D) which binds to theactive compound (part structure B) by way of the part structure C andinactivates this active compound by means of this binding.

[0149] Preferably, those nucleic acid sequences are used for partstructure D which encode part structure D in the naturally occurringprecursors (protein BSD), with part structure S being the naturalcleavage sequence in protein BSD.

[0150] The structures of the naturally occurring precursors of activecompounds (protein B) have already been reviewed, for example by Bartettet al., Mammalian Proteases, Academic Press, London (1980) in the caseof coagulation factors, complement factors and kallikrein, by Callard etal., The Cytokine Facts Book, Academic Press (1994) in the case ofinterleukins, chemokines and growth factors, and by Denhardt et al.,Pharmac. Ther. 59, 329 (1993) in the case of tissue inhibitors ofmetalloproteinases (TIMPs).

[0151] When selecting active compounds which do not have any naturallyoccurring precursors, and in the case of xenogeneic active compounds,use should be made of nucleic acid sequences, as component d), whichencode any peptide, preferably, however, of nucleic acid sequences whichencode those part structures D which naturally occur in the precursorsof human active compounds.

[0152] In order to facilitate secretion of the protein BCD, or B′BCD,which is expressed by the novel nucleic acid sequence, the homologoussignal sequence which may be present in the DNA sequence of component b)can be replaced with a heterologous signal sequence which improvesextracellular secretion. Thus, for example, the signal sequence forimmunoglobulin [DNA positions ≦63 to ≧107; Riechmann et al., Nature 332,323 (1988)] or the signal sequence for CEA [DNA positions ≦33 to ≧134,Schrewe et al., Mol. Cell Biol. 10, 2738 (1990); Berling et al., CancerRes. 50, 5634 (1990)] or the signal sequence of human respiratorysyncytial virus glycoproteins [cDNA of amino acids ≦38 to ≧50 or 48 to65; Lichtenstein et al., J. Gen. Virol. 77, 109 (1996)] can be inserted.

[0153] In addition, in order to augment translation, the nucleotidesequence GCCACC or GCCGCC [Kozak, J. Cell Biol. 108, 299 (1989)] can beinserted at the 3′ end of the promoter sequence and directly at the 5′end of the start signal (ATG) of the signal sequence.

[0154] Preparation of the Novel Nucleic Acid Constructs

[0155] The novel nucleic acid constructs which have been described areprepared by linking the individual components to each other usingstandard molecular biological methods.

[0156] Applications:

[0157] The novel nucleic acid construct is particularly well suited fortreating diseases which are accompanied by an increased local formationof proteases, such as tumor diseases, leukemias, allergies, autoimmunediseases, infections, inflammations, transplant rejection reactions,thromboses and blood vessel occlusions and other disturbances of bloodclotting and of blood circulation, and tissue injuries, includinginjuries to the central nervous system and damage to the nervous system.Administration is effected locally (e.g. onto the skin), nasally,orally, gastrointestinally, intrabronchially, intravesically,intravaginally, into the uterus, sub-cutaneously, intramuscularly,periarticularly, intraarticularly, into the cerebrospinal fluid, intothe brain tissue, into the spinal medulla, into wounds,intraperitoneally or intrapleurally, or systemically, e.g.intravenously, intraarterially, intraportally or into the heart.

[0158] In general, the administered composition comprises, whereappropriate in addition to the customary additives and auxiliarysubstances, either the novel nucleic acid construct or a cell which isable to express the novel nucleic acid construct. The administeredcomposition can be administered for the prophylaxis or therapy of adisease, as already described in detail above.

[0159] For administration purposes, an effective amount is determined bythe skilled artisan considering variables well known in the art such asthe nature of the applicable disease or condition, the nature of thepatient, mammal or cells being treated and the method of administration.

[0160] Moreover, in addition to the methods of administration discussedabove, the present invention contemplates the administration of thenovel nucleic acid construct to a mammal by ex vivo gene transfer of thecells of the subject mammal in a clinical setting. Such techniques arewell known to those of skill in the art. In addition, the presentinvention contemplates introduction of the novel nucleic acid constructinto cells in vivo [Rosenberg et al., Science 242:1575-1578 (1988) andWolff et al., PMAS 86:9011-9014 (1989)]. In this regard, the routes ofdelivery include systemic administration and administration in situ.Well-known techniques include systemic administration with cationicliposomes, and administration in situ with viral vectors. Any one of thegene delivery methodologies described in the existing art is suitablefor the introduction of novel nucleic acid construct into a target cell.

[0161] Said cell is prepared, for example, by transforming ortransfecting cells with the novel nucleic acid construct using methodsknown to the skilled person.

[0162] Examples of suitable cells are endothelial cells, lymphocytes,macrophages, glia cells, fibroblasts, liver cells, kidney cells, musclecells, cells of the bone or cartilage tissue, synovial cells, peritonealcells, skin cells, epithelial cells, leukemia cells and/or tumor cells.

[0163] The novel cells are also suitable for preparing the protein whichis encoded by the novel nucleic acid construct and which can be useddirectly as a drug.

[0164] The present invention furthermore relates, therefore, to the useof the novel nucleic acid construct for preparing a recombinantlyaltered cell, with the nucleic acid construct being introduced into thecells, to the use of the novel nucleic acid construct for preparing aprotein which is encoded by the nucleic acid construct, with the nucleicacid construct being caused to express in a suitable cell and theprotein which is formed being isolated, and to a cell which harbors thenovel nucleic acid construct. The above-described cells are thepreferred cells.

[0165] The following selection can, for example, be made from theabove-mentioned examples of promoter sequences and structural genes (forthe protein BCD or B′BCD) depending on the nature and site of thedisease and on the target cell to be transduced:

[0166] Therapy of Tumors:

[0167] Promoters [component a)]: endothelial cell-specific and cellcycle-specific or cell-nonspecific or muscle cell-specific and cellcycle-specific or tumor cell-specific (solid tumors, leukemias)

[0168] Ligands for the following target cells [component b′)]:proliferating endothelial cells or stroma cells and muscle cellsadjacent to the endothelial cell or tumor cells or leukemia cells.

[0169] Structural genes [component b)c)d)]: for coagulation-inducingfactors, for complement factors, for angiogenesis inhibitors, forcytostatic and cytotoxic proteins, for inducers of inflammations or forenzymes for activating precursors of cytostatic agents, for example forenzymes which cleave inactive precursor substances (prodrugs) therebyforming active cytostatic agents (drugs).

[0170] Therapy of Autoimmune Diseases and Inflammations:

[0171] Promoters [component a)]: endothelial cell-specific and cellcycle-specific, or macrophage-specific and/or lymphocyte-specific and/orcell cycle-specific or synovial cell-specific and/or cellcycle-specific.

[0172] Ligands for the following target cells [component b′)]:proliferating endothelial cells, macrophages and/or lymphocytes orsynovial cells.

[0173] Structural genes [component b)c)d)]: for the therapy ofantibody-mediated autoimmune diseases, for inhibitors of cellproliferation, cytostatic or cytotoxic proteins, enzymes for activatingprecursors of cytostatic agents or for the therapy of arthritis.

[0174] Therapy of Damage to the Nervous System:

[0175] Promoters [component a)]: glia cell-specific, endothelialcell-specific and cell cycle-specific or nonspecific and cellcycle-specific.

[0176] Ligands for the following target cells [component b′)]: gliacells or proliferating endothelial cells

[0177] Structural genes [component b)c)d)]: for neuronal growth factors,for example for cytokines and cytokine inhibitors which inhibit orneutralize the neurotoxic effect of TNFα.

[0178] Therapy of disturbances of the blood coagulation system and theblood circulation system:

[0179] Promoters [component a)]: cell-nonspecific, cell-nonspecific andcell cycle-specific or specific for endothelial cells, smooth musclecells or macrophages, or specific for endothelial cells, smooth musclecells or macrophages and cell cycle-specific.

[0180] Ligands for the following target cells [component b′)]:endothelial cells, proliferating endothelial cells or somatic cells inthe vicinity of endothelial cells and smooth muscle cells ormacrophages.

[0181] Structural genes [component b)c)d)]: for the inhibition ofcoagulation or for the promotion of fibrinolysis, for angiogenesisfactors, for hypotensive peptides, for an antiproliferative, cytostaticor cytotoxic protein or for an enzyme for cleaving is precursors ofcytostatic agents, thereby forming cytostatic agents, for inhibition ofthe proliferation of smooth muscle cells following injury to theendothelial layer or for blood plasma proteins, such as C1 inactivator,serum cholinesterase or α1-antitrypsin.

[0182] Therapy of Chronic Infectious Diseases:

[0183] Promoters [component a)]: virus-specific, cell-specific orvirus-specific or cell-specific and cell cycle-specific.

[0184] Ligands for the following target cells [component b′)]: livercells, lymphocyte and/or macrophage, epithelial cell or endothelialcell.

[0185] Structural genes [components b) c) d)]: for a protein whichexhibits cytostatic or cytotoxic effects, an enzyme which cleaves aprecursor of an antiviral or cytotoxic substance thereby forming theactive substance, or for antiviral proteins such as antivirally activecytokines and growth factors.

[0186] The invention is explained in more detail with the aid of thefollowing examples and figures without restricting it thereto:

EXAMPLES 1. Preparation of a Nucleic Acid Construct EncodingProstate-Specific Antigen (PSA)-Activatable FX

[0187] This deals with the preparation of a therapeutic agent fortreating prostate carcinoma metastases. Despite the surgical removal ofa prostate which has become carcinomatous, metastases of the prostatecarcinoma frequently arise which are currently still largely untreatableand which lead to the death of the patient. Such prostate carcinomametastases induce angiogenesis. Furthermore, prostate carcinomametastases secrete a tissue-specific enzyme, i.e. prostate-specificantigen (PSA). In accordance with the invention, a nucleic acidconstruct is prepared which, having been introduced into proliferatingendothelial cells, leads to a modified FX coagulation factor beingexpressed. The modification comprises replacing, in the gene for thenatural FX, the nucleotide sequence for the natural cleavage site, whosecleavage results in coagulation-active FXa, with a nucleotide sequenceencoding a PSA-specific cleavage site. As a result, the PSA which issecreted by prostate carcinoma metastases is able to specificallyactivate the modified FX which is secreted by proliferating endothelialcells in the vicinity of the metastases and thereby to initiate thecoagulation which leads to the blood supply to the metastasis beinginterrupted and consequently to necrosis of the metastasis.

[0188] The nucleic acid construct for the PSA-activatable FX is preparedin accordance with a scheme which is depicted in FIG. 3.

[0189] The DNA sequences of the individual components are joinedtogether, in the 5′ to 3′ direction, as follows:

[0190] Component a), which contains the promoter sequence of the cdc25Cgene [nucleic acids: −290 to +121; Lucibello et al., EMBO J. 14, 132(1995); Zwicker et al., Nucl. Acids Res. 23, 3822 (1995); EMBO J. 14,4514 (1995)], the sequence GCCACC (Kozak, J. Cell Biol. 108, 229 (1989))and the cDNA for the immunoglobulin signal peptide [nucleotide sequence≦63 to ≧107; Riechmann et al., Nature 332, 323 (1988)], is fused tocomponent b)c)d), which contains the cDNA for human FX (nucleotidesequence 1 to ≧1468) [Messier et al., Gene 99, 291 (1991)] in whichamino acid 194 has been mutated from Arg to Tyr.

[0191] The individual components of the construct are linked by way ofsuitable restriction sites which are introduced at the termini of thedifferent elements by way of PCR amplification. The linking is effectedusing enzymes which are specific for the restriction sites and which areknown to the skilled person, and DNA ligases. These enzymes can beobtained commercially.

[0192] The nucleotide construct which has been prepared in this way iscloned into pUC 18/19 or Bluescript-derived plasmid vectors.

2. Expression in Human Embryonic Kidney Cells

[0193] Proliferating human embryonic kidney cells [HEK 293; Racchi etal., J. Biol. Chem. 268, 5735 (1993)] which are being maintained inculture are transfected with the above-described plasmid using themethod known to the skilled person [Graham and van der Eb, Virol. 52,456 (1973)].

[0194] The mutated factor X is purified from the supernatant fromapprox. 10⁷ transfected HEK 293 cells [Watzke et al., J. Clin. Invest.88, 1685 (1991)] and assayed in a coagulation test for factor X with andwithout the addition of PSA. Purified PSA is obtained from Chemicon(Temecula, Calif., USA).

[0195] In this test, the coagulation defect in human FX-deficient plasmais counterbalanced by functionally active FXa.

[0196] Nonmutated (wild-type) FX (which is activated by Russel's vipervenom) is employed as a positive control. In addition to the testmixture lacking PSA, a mock preparation from the supernatant fromuntransfected HEK 293 cells is used as a negative control.

[0197] The coagulation activity of the mutated FX is measured byrecalcification time (Seitz R et al., Int. J. Cancer 53:514-520, 1993).100 μl of FX-deficient plasma (Behringwerke, Marburg) are incubated, at37° C. for 120 sec, with 100 μl of the FX preparation from the cellsupernatant. The FX preparation contains PSA as activator. No PSA isadded in the case of the negative control. FX (wild-type) and Russel'sviper venom (RVV) are employed as the positive control. The coagulationreaction is augmented by adding 100 μl of 0.02 M CaCl2 and determined ina coagulometer.

[0198] The following results are obtained:

[0199] The negative controls without any activation of coagulation givea coagulation time of approx. 200 sec. By contrast, significantlyshorter coagulation times, of 50 sec, are achieved when activated FX(mutated FX and PSA or wild-type FX and RVV) is used.

[0200] It can be concluded from this that the transduced HEK 293 cellsexpress mutated FX which, in the added presence of PSA, counterbalancesthe coagulation defect of FX-deficient plasma.

3. Expression in Human Endothelial Cells

[0201] Human umbilical cord endothelial cells which are being maintainedin culture are transfected with the above-described plasmid using themethod known to the skilled person (Lucibello et al., EMBO J. 14, 132(1995).

[0202] In order to check cell cycle specificity, endothelial cells aresynchronized in G0/G1 by withdrawing methionine over a period of 48hours. After staining with Hoechst 33258 (Hoechst AG, Frankfurt), theDNA content of the cells is determined in a fluorescent-activated cellsorter (Lucibello et al., EMBO J. 14, 132 (1995).

[0203] The expression of the nucleic acid construct is assayed in thesupernatant from the endothelial cells in analogy with the investigationcarried out on the HEK 293 cells.

[0204] The following results are obtained:

[0205] The protein which is expressed by the transfected endothelialcells counterbalances the coagulation defect of FX-deficient plasma, incontrast to mock preparations from the supernatant from untransfectedendothelial cells.

[0206] A markedly higher concentration of mutated FX can be detected inthe supernatant from proliferating, transduced endothelial cells(DNA>2S) as compared with the supernatant from endothelial cells whichhave been synchronized in G0/G1 (DNA=2S).

[0207] Consequently, the above-described nucleic acid construct leads tothe gene for the mutated FX being expressed in a cell cycle-dependentmanner in endothelial cells, and this mutated FX can be activated by PSAsuch that it brings about coagulation in FX-deficient plasma.

[0208] Federal Republic of Germany priority application, DE 19701141.1,filed Jan. 16, 1997, including the specification, drawings, claims andabstract, is hereby incorporated by reference.

1 62 4 amino acids amino acid <Unknown> linear peptide 1 Xaa Xaa Arg Xaa1 68 base pairs nucleic acid double linear DNA 2 AGCAGGTGTT GGGAGGCAGCAGGTGTTGGG AGGCAGCAGG TGTTGGGAGG CAGCAGGTGT 60 TGGGAGGC 68 41 base pairsnucleic acid double linear DNA 3 GGCCGATGGG CAGATAGAGG GGGCCGATGGGCAGATAGAG G 41 5 amino acids amino acid <Unknown> linear peptideinternal 4 Cys Pro Gly Arg Val 1 5 6 amino acids amino acid <Unknown>linear peptide internal 5 Cys Pro Gly Arg Val Val 1 5 5 amino acidsamino acid <Unknown> linear peptide internal 6 Cys Pro Gly Arg Ile 1 5 6amino acids amino acid <Unknown> linear peptide internal 7 Cys Pro GlyArg Ile Val 1 5 4 amino acids amino acid <Unknown> linear peptideinternal 8 Pro Arg Phe Lys 1 5 amino acids amino acid <Unknown> linearpeptide internal 9 Pro Arg Phe Lys Ile 1 5 6 amino acids amino acid<Unknown> linear peptide internal 10 Pro Arg Phe Lys Ile Ile 1 5 6 aminoacids amino acid <Unknown> linear peptide internal 11 Pro Arg Phe LysIle Val 1 5 5 amino acids amino acid <Unknown> linear peptide internal12 Arg Arg Phe Phe Leu 1 5 6 amino acids amino acid <Unknown> linearpeptide internal 13 Arg Arg Phe Phe Leu His 1 5 6 amino acids amino acid<Unknown> linear peptide internal 14 Arg Arg Phe Phe Leu Val 1 5 5 aminoacids amino acid <Unknown> linear peptide internal 15 Arg Arg Phe PheIle 1 5 6 amino acids amino acid <Unknown> linear peptide internal 16Arg Arg Phe Phe Ile His 1 5 6 amino acids amino acid <Unknown> linearpeptide internal 17 Arg Arg Phe Phe Ile Val 1 5 8 amino acids amino acid<Unknown> linear peptide internal 18 Ser Phe Ser Ile Gln Tyr Ile Val 1 58 amino acids amino acid <Unknown> linear peptide internal 19 Gly SerGln Gln Leu Leu Ile Val 1 5 8 amino acids amino acid <Unknown> linearpeptide internal 20 Gly Ile Ser Ser Gln Tyr Ile Val 1 5 6 amino acidsamino acid <Unknown> linear peptide internal 21 Pro Arg Phe Lys Ile Ile1 5 6 amino acids amino acid <Unknown> linear peptide internal 22 ProArg Phe Lys Ile Val 1 5 4 amino acids amino acid <Unknown> linearpeptide internal 23 Lys Ser Arg Met 1 4 amino acids amino acid <Unknown>linear peptide internal 24 Lys Ser Arg Ile 1 4 amino acids amino acid<Unknown> linear peptide internal 25 Lys Met Arg Arg 1 4 amino acidsamino acid <Unknown> linear peptide internal 26 Lys Met Arg Ile 1 4amino acids amino acid <Unknown> linear peptide internal 27 Ile Arg ArgArg 1 4 amino acids amino acid <Unknown> linear peptide internal 28 IleArg Arg Ile 1 4 amino acids amino acid <Unknown> linear peptide internal29 Arg Ala Arg Leu 1 4 amino acids amino acid <Unknown> linear peptideinternal 30 Arg Ala Arg Ile 1 4 amino acids amino acid <Unknown> linearpeptide internal 31 Gln Ala Arg Phe 1 4 amino acids amino acid <Unknown>linear peptide internal 32 Gln Ala Arg Ile 1 4 amino acids amino acid<Unknown> linear peptide internal 33 Lys Leu Arg Leu 1 4 amino acidsamino acid <Unknown> linear peptide internal 34 Lys Leu Arg Ile 1 5amino acids amino acid <Unknown> linear peptide internal 35 Gly Gly GlyAla Gln 1 5 6 amino acids amino acid <Unknown> linear peptide internal36 Gly Gly Gly Ala Gln Leu 1 5 5 amino acids amino acid <Unknown> linearpeptide internal 37 Gln Leu Gly Val Met 1 5 6 amino acids amino acid<Unknown> linear peptide internal 38 Gln Leu Gly Val Met Gln 1 5 5 aminoacids amino acid <Unknown> linear peptide internal 39 Ala Ala Ala SerLeu 1 5 6 amino acids amino acid <Unknown> linear peptide internal 40Ala Ala Ala Ser Leu Lys 1 5 5 amino acids amino acid <Unknown> linearpeptide internal 41 Val Ala Val Ser Ala 1 5 6 amino acids amino acid<Unknown> linear peptide internal 42 Val Ala Val Ser Ala Lys 1 5 5 aminoacids amino acid <Unknown> linear peptide internal 43 Leu Ala Ala AsnLeu 1 5 6 amino acids amino acid <Unknown> linear peptide internal 44Leu Ala Ala Asn Leu Arg 1 5 5 amino acids amino acid <Unknown> linearpeptide internal 45 Gly Pro Gln Gly Ile 1 5 6 amino acids amino acid<Unknown> linear peptide internal 46 Gly Pro Gln Gly Ile Ala 1 5 5 aminoacids amino acid <Unknown> linear peptide internal 47 Gly Pro Gln GlyLeu 1 5 6 amino acids amino acid <Unknown> linear peptide internal 48Gly Pro Gln Gly Leu Leu 1 5 5 amino acids amino acid <Unknown> linearpeptide internal 49 Gly Pro Gln Gly Leu 1 5 6 amino acids amino acid<Unknown> linear peptide internal 50 Gly Pro Gln Gly Leu Ala 1 5 5 aminoacids amino acid <Unknown> linear peptide internal 51 Gly Ile Ala GlyIle 1 5 6 amino acids amino acid <Unknown> linear peptide internal 52Gly Ile Ala Gly Ile Thr 1 5 5 amino acids amino acid <Unknown> linearpeptide internal 53 Gly Leu Pro Gly Ile 1 5 6 amino acids amino acid<Unknown> linear peptide internal 54 Gly Leu Pro Gly Ile Gly 1 5 5 aminoacids amino acid <Unknown> linear peptide internal 55 Gly Phe Pro GlyIle 1 5 6 amino acids amino acid <Unknown> linear peptide internal 56Gly Phe Pro Gly Ile Gly 1 5 5 amino acids amino acid <Unknown> linearpeptide internal 57 Gly Pro Ala Gly Ile 1 5 6 amino acids amino acid<Unknown> linear peptide internal 58 Gly Pro Ala Gly Ile Ser 1 5 5 aminoacids amino acid <Unknown> linear peptide internal 59 Gly Pro Ala GlyIle 1 5 6 amino acids amino acid <Unknown> linear peptide internal 60Gly Pro Ala Gly Ile Ala 1 5 5 amino acids amino acid <Unknown> linearpeptide internal 61 Ser Gly Thr Glu Ile 1 5 6 amino acids amino acid<Unknown> linear peptide internal 62 Ser Gly Thr Glu Ile Val 1 5

What is claimed is:
 1. A nucleic acid construct for expressing an activesubstance which is activated by an enzyme which is released frommammalian cells, wherein said construct comprises the following nucleicacid sequences in the following order: a) at least one promoter elementoperably linked to; b) at least one nucleic acid sequence which encodesan active compound, wherein said active compound is endogenous tomammals, operably linked to; c) at least one nucleic acid sequence whichencodes an amino acid sequence cleavable specifically by an enzyme whichis released from a mammalian cell, operably linked to; d) at least oneDNA sequence which encodes a polypeptide which is bound to said activecompound by said cleavable amino acid sequence and inhibits the activityof said active compound, and wherein said nucleic acid component c) doesnot naturally occur as operably linking said nucleic acid sequence b) tosaid nucleic acid d).
 2. A nucleic acid construct as claimed in claim 1,wherein said enzyme is a protease.
 3. A nucleic acid construct asclaimed in claim 1, wherein said enzyme is a prostate specific antigen,a plasminogen activator, a cathepsin or a matrix metalloproteinase.
 4. Anucleic acid construct as claimed in claim 1, wherein said mammaliancells are tumor cells, leukemia cells, endothelial cells, macrophages,lymphocytes, muscle cells, epithelial cells, glia cells, synovial cellsor virus-infected cells.
 5. A nucleic acid construct as claimed in claim1, wherein said nucleic acid construct further comprises a nucleic acidsequence operably linked to said construct of claim 1, wherein saidnucleic acid sequence encodes a ligand which binds said active compoundto a target structure.
 6. A nucleic acid construct as claimed in claim1, wherein said nucleic acid sequences b) and d) of claim 1 encode partsof a natural precursor of a protein active compound, wherein the nucleicacid sequence encoding the cleavage sequence naturally occurring betweensaid nucleic acid sequences b) and d) has been replaced by said nucleicacid sequence c), which does not naturally occur between said nucleicacid sequences b) and d).
 7. A nucleic acid construct as claimed inclaim 1, wherein said polypeptide encoded by said nucleic acid sequenced) is part of a natural precursor of a protein active compound.
 8. Anucleic acid construct as claimed in claim 1, wherein said construct isoperably inserted into a plasmid or a viral vector.
 9. A nucleic acidconstruct as claimed in claim 1, wherein said nucleic acid sequence a)is a promoter sequence which can be activated nonspecifically,cell-specifically, virus-specifically, metabolically, cellcycle-specifically or by tetracycline.
 10. A nucleic acid construct asclaimed in claim 1, wherein said nucleic acid sequence a) comprises atleast two identical or two different promoter sequences.
 11. A nucleicacid construct as claimed claim 9, wherein said nucleic acid sequence a)is activated in endothelial cells, in cells adjoining activatedendothelial cells, in muscle cells, in leukemia cells, in tumor cells,in glia cells, in lymphocytes, in macrophages or in synovial cells. 12.A nucleic acid construct as claimed in claim 1, wherein said activecompound activates or inhibits a biological activation cascade or is anactive component of this cascade, or activates or inhibits thecoagulation system, activates fibrinolysis, activates the complementsystem or activates the kinin system, or is an enzyme which converts theinactive precursor of a pharmacological substance into the activesubstance, or which itself is a pharmacologically active substance. 13.A nucleic acid construct as claimed in claim 12, wherein said activecompound is a coagulation factor which is selected from the groupconsisting of thrombin, factor Va, factor VIIa, factor IXa, factor Xa,TF coagulation-active fragments or factor XIIa; thrombin which ismutated in the region of the Arg-Thr cleavage site (amino acid position327/328); a fibrinolytic protein which is selected from urokinase, tPAor functional hybrids thereof; a complement factor which is selectedfrom CVF, C3b or functional cleavage products thereof; an antithromboticprotein which is selected from protein C, C-1S inhibitor,α1-antitrypsin, hirudin, AT-III, TFPI, PAI-1, PAI-2 or PAI-3; akallikrein; a cytostatic, cytotoxic or inflammation-eliciting protein;an antiangiogenic protein; an immunomodulatory protein; anantiinflammatory protein; a protein which relieves damage to the nervoussystem; a protein which inhibits or neutralizes the neurotoxic effect ofTNFα; an angiogenesis-stimulating protein; a hypotensive protein; anantiviral protein; a cytokine; an interferon; a tumor necrosis factor;oncostatin M or LIF; a cytokine receptor; the moiety of a cytokinereceptor which is external to the cell; a cytokine antagonist; a growthfactor; a growth factor receptor; the moiety of a growth factor receptorwhich is external to the cell; a chemokine; angiostatin; platelet factor4; TIMP-1, TIMP-2 or TIMP-3; a nitroreductase; a β-glucuronidase; acarboxypeptidase; a β-lactamase; a cytosine deaminase; a catalase; aperoxidase; a phosphatase; an oxidase; kallikrein or an endothelial cellnitric oxide synthase.
 14. A nucleic acid construct as claimed in claim1, which further comprises a nucleic acid sequence b′) which encodes aligand which binds to a cell membrane receptor, a cell membrane antigen,a cell membrane-located adhesion molecule, or to the extracellularmatrix or component thereof.
 15. A nucleic acid construct as claimed inclaim 14, wherein said ligand is an antibody or an antibody fragmentwhich binds specifically to a cell membrane antigen or to an antigen onthe extracellular matrix, or is a polypeptide which binds to receptor onthe cell membrane wherein said polypeptide is a growth factor, acytokine, an interferon, a tumor necrosis factor, a chemokine, areceptor-binding part sequence of these ligands, a peptide hormone,angiotensin, kinin, folic acid, an adhesion molecule or the partsequence of the adhesion molecule which binds to the correspondingadhesion molecule or to the extracellular matrix, an extracellularmoiety of an Fc receptor, a glycoprotein of a virus, a part sequence ofthe glycoprotein which binds to these cells, the transmembrane domain ofa receptor or of a viral glycoprotein, or a glycophospholipid anchor.16. A nucleic acid construct as claimed in claim 14, wherein said ligandbinds to activated or proliferating endothelial cells, to tumor cells,to muscle cells, preferably smooth muscle cells, to fibroblasts, tomacrophages, to lymphocytes, to liver cells, to kidney cells, tosynovial cells, to inflammatory cells, to virus-infected cells, tobronchial epithelial cells, to glia cells or to leukemia cells.
 17. Anucleic acid construct as claimed in claim 14, wherein the constructcomprises at least two identical or different nucleic acid sequencesb)c)d) or b′)b)c)d), which nucleic acid sequences are linked to eachother by way of an internal ribosomal entry site.
 18. A process forpreparing a nucleic acid construct according to claim 1, which comprisesoperably linking said nucleic acid sequences of claim
 1. 19. A methodfor the treatment or prophylaxis of tumors, leukemias, allergies,autoimmune diseases, infections, inflammations, transplant rejectionreactions, thromboses, blood vessel occlusions, blood coagulation, bloodcirculation disturbances, injuries to tissues, or damage to the nervoussystem, comprising administering to a mammal an effective amount of apolypeptide expressed by the nucleic acid construct of claim
 1. 20. Amethod for preparing a recombinantly altered cell, comprisingtransducing a suitable cell with said nucleic acid construct of claim 1.21. A method for preparing a polypeptide which is encoded by saidnucleic acid construct of claim 1, comprising transducing a suitablecell with said construct, expressing said polypeptide in said cell, andisolating said expressed polypeptide.
 22. The method of claim 20,wherein said cell is an endothelial cell, a lymphocyte, a macrophage, aglia cell, a fibroblast, a liver cell, a kidney cell, a muscle cell, acell of the bone or cartilage tissue, a synovial cell, a peritonealcell, a skin cell, an epithelial cell, a leukemia cell or a tumor cell.23. The method of claim 21, wherein said cell is an endothelial cell, alymphocyte, a macrophage, a glia cell, a fibroblast, a liver cell, akidney cell, a muscle cell, a cell of the bone or cartilage tissue, asynovial cell, a peritoneal cell, a skin cell, an epithelial cell, aleukemia cell or a tumor cell.
 24. A cell transduced with said nucleicacid construct of claim
 1. 25. A protein encoded by said nucleic acidconstruct of claim 1.